Fig. 4: Peripheral lysosomal bias leads to enhanced leader cell emergence in collectively migrating epithelia. | Nature Communications

Fig. 4: Peripheral lysosomal bias leads to enhanced leader cell emergence in collectively migrating epithelia.

From: Mechanosensitive dynamics of lysosomes along microtubules regulate leader cell emergence during collective cell migration

Fig. 4: Peripheral lysosomal bias leads to enhanced leader cell emergence in collectively migrating epithelia.The alternative text for this image may have been generated using AI.

A Representative confocal micrograph of control and kinesore treated MDCK cells, migrated for T-2 h post confinement lift-off. Yellow arrowhead marks the leader cell. B Dot-column plot of POF-lysosomes in control (n = 101), and kinesore (n = 53) treated cells (3 independent experiments). Unpaired Student’s t-test with Welch’s correction (two-tailed) performed. C Mouse embryonic skin treated with kinesore subjected to wounding and migration post-incision on an air-liquid interface with the media containing the inhibitor. Yellow arrowheads mark lysosome enrichment in cells at the leading edge. Pink arrowhead marks the absence of lysosome polarization on kinesore treatment. Presence of enriched actin cables observed in kinesore treated mouse embryonic skin wounds at the migration front, indicating a lack of migratory cells. D Dot-column plot of POF-lysosomes in control (n = 144), and kinesore (n = 165) treated mouse embryonic skin wounds (3 independent experiments). Unpaired Student’s t-test with Welch’s correction (two-tailed). E–H Schematic and confocal micrographs of molecular basis and lysosome positioning bias to cell periphery (E, F) and cell center (G, H) using the RAMP system. I Fluorescence/bright-field time-lapse imaging snapshots of MDCK cells stably expressing mCherry-Kif5B*-Strep transiently transfected with LAMP1-SBP-GFP. Yellow arrowheads mark co-expression. J Time-lapse imaging snapshots of MDCK cells expressing Strep-KifC1*-mCherry transiently transfected with LAMP1-SBP-GFP. Yellow arrowheads mark co-expression. K Percentage of leader cells emerging in cells co-expressing mCherry-Kif5B*-Strep or Strep-KifC1*-mCherry and LAMP1-SBP-GFP (n = 12), Tom20-SBP-GFP (n = 28), and VAPA-SBP-GFP (n = 28) migrated for 4h (n - number of ROIs analysed, each ROI has multiple transfected cells, data shown is the average leader cell emergence percent from 3 independent experiments). One-Way ANOVA test. L MDCK cells co-expressing either mCherry-Kif5B*-Strep or Strep-KifC1*-mCherry and LAMP1-SBP-GFP, fixed at T-2 h and stained for focal adhesion protein Paxillin. M LAMP1-SBP-GFP and mCherry-Kif5B*-Strep or Strep-KifC1*-mCherry double-positive cells were imaged live for 24 h and quantified for the fraction of leader cells emerging. Inverted gray-scale images of cells fixed post-migration and stained for actin. n (ROIs) = 20 per condition, 3 independent experiments. Student’s t-test, unpaired, two-tailed. N Percentage of leader emergence in MDCK cells co-expressing KLC2 and SKIP-WT (n = 35) or SKIP WD- > A (n = 32) mutant and KLC2 alone (n = 37) (3 independent experiments). One-Way ANOVA test. Scale Bars-10 µm. Data are mean ± s.e.m.

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